This invention related to a system for stably expressing a low-Km cAMP phosphodiesterase (PDE) and the use of the system for assessing inhibitors of PDE. In particular, this invention related to a system for the stable expression of a low-Km PDE IV and the use of the stable system in the evaluation of inhibitors of PDE IV. This invention also relates to an improved in vitro assay for the evaluation of inhibitors of PDE IV directed against a "high-affinity" state enzyme with respect to rolipram.
Phosphodiesterases (PDEs) are a family of enzymes that metabolize 3',5' cyclic nucleotides to inactive metabolites, thereby terminating their second messenger role in mediating the cellular responses to various hormones and neurotransmitters. To date, seven families of PDE enzymes have been identified and each isoenzyme has been mapped to a distinct gene locus. These isoenzymes exhibit different substrate specificities for cAMP and/or cGMP, catalytic activities, tissue and cellular distributions, and sensitivity to different endogenous activators and inhibitors. In addition, selective inhibitors of some of these isoenzymes have been synthesized, and have been validated as useful tools for examining the biological function(s) of these enzymes in various tissues (See reference (1)).
Recently, the low-Km cAMP specific, type IV PDE family of enzymes has generated considerable interest as potential targets for the development of novel antiasthmatic and antiinflammatory drugs (2). Within this family of PDEs exist at least four isoenzymes, each of which is encoded by a distinct gene (3). Additionally, the mRNA of each gene product is thought to undergo alternative splicing, thereby giving rise to isoforms for each isoenzyme, respectively (4,5). The four known PDE IV gene products (a,b,c,d) exhibit different tissue distributions (6), but in general are expressed in a wide number of cells that play a role in allergic and inflammatory responses (7). Investigators have utilized rolipram and Ro 20-1724, both potent and selective prototypic inhibitors of the PDE IV enzyme, to examine the effect of PDE IV inhibition on the activation state of inflammatory cells. Inhibitors of PDE IV enzyme(s) have been shown to block antigen-induced airway eosinophilia in allergic guinea pigs (8), inhibit superoxide production by human neutrophils (9), suppress LPS induction of TNFa release from human monocytes (10), inhibit arachidonate release from human neutrophils (11) and to block ozone-induced airways hyperreactiviy in a variety of species (12).
To date, the potentially attractive antiasthmatic and antiinflammatory properties of drugs that inhibit PDE IV enzymes have been offset by their ability to cause gastrointestinal discomfort (13), and CNS hyperactivity (14). More importantly, however, the potency of this class of drug appears to track with its propensity to cause nausea and vomiting in the clinical setting. These dose limiting side effects have precluded the clinical development of many potent and potentially therapeutically useful PDE IV enzyme inhibitors.
In addition to the pronounced clinical side effects exhibited by PDE IV inhibitors, problems exist with the mechanistic basis by which these compounds are thought to act. Alterations in cellular function by these compounds are expected to be accompanied by increases in the cellular content of cAMP. Examples have been reported in the literature demonstrating a lack of correlation between elevations in cAMP and endpoint cellular measurements (16,17,18). Additionally, the potency of these compounds in elevating cellular cAMP in the target cell does not always translate biochemically into their ability to inhibit the PDE IV enzyme in a broken cell preparation.
Herein we report the establishment and characterization of a CHO-K1 cell line which stably expresses high levels of a full length low-Km cAMP specific PDE IVa enzyme. We have examined several well known potent PDE IV enzyme inhibitors and have compared the rank order of their potencies in elevating cAMP in a whole-cell preparation with their ability to inhibit phosphodiesterase activity in a broken-cell preparation.
We have surprisingly found that the soluble enzyme inhibition assay described in the prior art does not reflect behavior of the inhibitors acting in vivo. We have developed an improved soluble enzyme assay which is superior in terms of it ability to reflect the behavior of inhibitors acting in vivo. We have also developed a the whole-cell assay which reflects the behavior of inhibitors acting in vivo.